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New Fish Virus Characterized in Genome Sequencing Study

NEW YORK (GenomeWeb) – An international team has identified a new orthomyxo-like virus that appears to have contributed to large tilapia fish die-offs in the Middle East and South America over the past few years.

As they reported in mBio today, researchers from the US, Israel, Scotland, and the West Indies used sequencing, mass spec, and other approaches to characterize the newly discovered tilapia lake virus (TiLV), which resembles orthomyxoviruses such as the influenza C virus, the infectious salmon anemia orthomyxovirus, and the Thogoto virus. Their experiments support the notion that TiLV, a negative strand RNA virus with a 10-gene genome, is capable of infecting fish cells, producing pathogenic features resembling those in infected tilapia.

"The more we studied them, the more convinced we became that what we had represented an entirely new virus," senior author Ian Lipkin, director of Columbia University's Center for Infection and Immunity, said in a statement.

Lipkin noted that analyses of the newly identified virus should help in detecting TiLV in infected tilapia stocks and developing vaccines against it to safeguard the tilapia trade — an industry with an estimated value of around $7.5 billion each year — and to ensure the availability of a relatively affordable protein source in parts of the developing world.

Both wild and commercially farmed tilapia in Israel's Sea of Galilee (Kinneret Lake), have experienced multiple disease outbreaks and die-offs since 2009, with fish succumbing to skin, kidney, nervous system problems. Similar die-offs have since occurred at Nile tilapia farms in Ecuador, though the affected fish tissues differed somewhat compared to infected fish in Israel.

An initial search for an infectious culprit in affected fish from Israel, published by Israeli researchers in the Journal of Clinical Microbiology in 2014, pointed to the presence of an unknown RNA virus dubbed TiLV in infected fish. And in unpublished PCR-based tests, Lipkin and others uncovered the same virus was behind the South American outbreaks.

For the new analysis, Lipkin and his colleagues used a combination of Ion Torrent and Illumina sequencing technologies to generate sequences from ribosomal RNA-depleted, concentrated viral particle-containing samples obtained from an infected snakehead fish cell line called E-11.

After filtering out messenger RNA sequences from the host fish and RNA associated with known viruses in sequence databases, the team put together a TiLV genome using the remaining viral reads.

The 10 contig clusters in this genome each appear to code for a different protein, the researchers found. Nine of the 10 open reading frames did not show homology to known ORFs, they noted, while the PB1-coding segment shared some similarities to the PB1 sequence described in the influenza C virus.

When the team re-sequenced isolates from Ecuadorian tilapia that had tested positive for TiLV by PCR, it found that the TiLV isolates from that country shared between 97 percent and 99 percent nucleotide sequence identity with the original TiLV genome.

Consistent with its suspected negative strand RNA genome composition, the researchers' experiments indicated that TiLV could infect fish cells when viral proteins were present. In contrast, de-proteinized TiLV did not produce characteristic cytopathic features in infected E-11 cells. Further, the researchers used in situ hybridization to demonstrate that TiLV was present in symptomatic liver or brain tissues from infected fish.

"The presence of viral nucleic acid at sites of pathology in brain and liver together with the observation that virus propagated in cell culture is capable of inducing disease in naïve fish implicates TiLV as the causative agent of outbreaks of both viral encephalitis and syncytial hepatitis in tilapiines in Israel and Ecuador," the authors concluded.